8
D. S.BERNS,H. L. CRESPIAND J. J. KATZ
and succinate ester^.^^" A possible explanation for this difference could reside in the preferential conformation of the ground states for the dimethylamines and the carboxylic acids. Possibly owing to the solvation of the carboxyl anion the glutarate and succinate monoesters exist preferentially in an extended conformation, whereas the relatively non-polar amines exist in a coiled conformation due to formation of hydrophobic bonds (Chart 11). I n this connection i t should be noted that rate studies conducted in 50yodioxane-water and also in 8 M aqueous urea solution failed to alter this ratio. 37 (37) These experiments are probably not critical since the uncoiling of hydrocarbon chains due to clathrate formation requires at least a 7-membered chain (see L. F. Fieser and M. Fieser, “Advanced Organic Chemistry,’’ Reinhold Publishing Corp., New York, N. Y.,1961, p p . 131-133). Also, the water content of 507, dioxane-water would probably not be low enough t o bring about uncoiling.
[CONTRIBUTION FROM THE CHEMISTRY DIVISION,
Vol. 85
A second explanation would implicate steric hindrance imposed by the dimethyl substitution of the amino group on the rate of closure to form the five-membered ring (though the magnitude of the rate constants would not appear to suggest any steric hindrance; see Table VII). I t should be noted that the ratio of the rate constants for the formation of five- and six-membered rings in the cyclization of w-aminoalkyl bromides in water is 800.3s Our investigations in this area are continuing. Acknowledgments.-This work was supported by grants from the National Science Foundation and the National Institutes of Health. S. J. Benkovic particularly acknowledges support in the nature of a predoctoral fellowship from the National Institutes of Health. (38) G. Solomon, Helv. Chim. Acta, 16, 1361 (1933); 17, 851 (1934).
ARGONNE NATIONAL LABORATORY,
ARGONNE,
ILL.]
Isolation, Amino Acid Composition and Some Physico-chemical Properties of the Protein Deuterio-phycocyanin1 BY DONALD S. B E R ~ ’ sHENRY ,~ L. CRESPIAND
JOSEPH
J. KATZ
RECEIVED AUGUST15, 1962 The isolation and purification of a fully deuteriated protein, deuterio-phycocyanin, from blue-green algae grown autotrophically in 99.8y0 D20 is described. Sedimentation behavior in the ultracentrifuge shows the protein to be a system of reversibly interacting components. The amino acid compositions of ordinary and deuteriophycocyanin isolated from Plectonema calothricoides have been established and it appears that the amino acid compositions are identical within experimental error. Ordinary and deuterio-phycocyanin therefore probably differ only in isotopic composition. Thermal denaturation of ordinary and deuterio-phycocyanin, both dissolved in H20, has been studied by measuring the quenching of fluorescence. Deuterio-phycocyanin undergoes thermal denaturation a t a temperature 5’ lower than is the case for ordinary phycocyanin. Since the two proteins appear t o differ primarily in the isotopic composition of the non-polar side chains, differences in denaturation behavior are probably to be ascribed to differences in hydrophobic bonding.
Introduction The successful cultivation of fully deuteriated organisms on the large scale by Katz, Crespi and coworkers3 has made a great variety of fully deuteriated substances accessible for the first time. Proteins in which hydrogen has been entirely replaced by deuterium may be expected to make a useful contribution to problems of protein structure and function, and efforts have therefore been directed to the preparation of such substances. In the present communication the isolation, purification, amino acid composition and behavior on thermal denaturation of the fully deuteriated protein phycocyanin, extracted from the fully deuteriated bluegreen alga Plectonema calothricoides, are described ; a preliminary description of some aspects of this work has already a ~ p e a r e d . ~ Phycocyanin is a blue photosynthetic pigment widely distributed in blue-green algae. A member of the class of biliproteins, for which molecular weights of the order of 200,000 to 300,000 have been quoted, phycocyanin appears in fact to be a system of reversibly interacting components. The chemical properties of phycocyanin are reviewed by O’hEocha.6 For purposes of the present discussion the ordinary, hydrogen-containing phycocyanin extracted from algae grown in (1) Based on work performed under the auspices of the U. S. Atomic Energy Commission. Presented in part at the 141st National Meeting of the American Chemical Society in Washington, D. C., April, 1962. (2) Resident research associate, 1861-1962. (3) H. DaBoll, H . L. Crespi and J. J. Katz, Biofechnologr and Bioengineering, to be published. (4) D. S. Berns, H . L. Crespi and J. J. Katz, J . Am. Chem. Soc., 84, 486 (1962). (5) C. O’hEocha, in “Comparative Biochemistry of Photoreactive Systems,” M. B. Allen, ed., Academic Press, Inc., New York, N. Y.,1860, pp.
181-205.
HzO will be referred to as ordinary or protio-phycocyanin; the protein extracted from algae grown in 99.8% DaO will be designated deuterio-phycocyanin. The prefix deuterio- will also be used when the deuteriophycocyanin is dissolved in HzO and the exchangeable positions of the protein are occupied by hydrogen. The sharp distinction between the protein described here, in which the non-exchangeable positions in the molecule are occupied by deuterium, and ordinary proteins into which deuterium is introduced into exchangeable positions by treatment with Dz06 can be readily inferred from the discussion of Scheraga’ on hydrogen-deuterium exchanges in proteins. Experimental Isolation and Purification .-Deuterio-phycocyanin and protiophycocyanin were isolated from the blue-green alga Plectonema calothricoides by either of two procedures. In the first, approximately 25 g. of algae (wet weight) were frozen and thawed twice to rupture the cells. About 200 ml. of aqueous acetate buffer (pH 4.7, p = 0.1) was added and the solution allowed to stand, first for several hours a t room temperature, and then in the refrigerator a t 5’. After several days, the supernatant solution was quite blue and intensely fluorescent. The supernatant solution was removed by centrifugation, fresh buffer was added, and the extraction continued until most of the phycocyanin was removed from the algae. An olive-green appearance of the residue indicated that most of the blue pigment had been extracted. The aqueous extract was then centrifuged for 10 minutes a t 4’ a t 12,000 r.p.m. to remove debris. Phycocyanin was then precipitated from the clarified extract by adding ammonium sulfate to 50y0 saturation. The second procedure for the extraction of phycocyanin from algae omitted freezing and thawing. Instead, cell lysis was achieved by the action of lysozyme (Worthington 2 >
